Structure-Activity Relationship (SAR) studies provide valuable insights for understanding intermolecular interactions between bioactive molecules. In their natural states, bioactive molecules often adopt unique, conformationally-constrained structures in order to recognize and bind to their binding partners, to form a molecular complex therewith, and in turn to elicit specific activities. In particular, protein-protein interactions are crucial events involved in most biological and pathological processes, and are therefore logical targets for drug design. Important protein-protein interactions occur between such binding partners as enzyme-substrate, ligand-receptor, and antigen-antibody complexes.
One of the revolutionary advances in drug discovery is the development of combinatorial libraries. Combinatorial libraries are collections of different molecules, such as peptides, that can be made synthetically or recombinantly. Member peptides in a combinatorial peptide library include amino acids incorporated randomly into certain or all positions of their sequences. Such libraries have been generated and used in various ways to screen for peptide candidates which bind effectively to target molecules and to identify such sequences.
Many methods for generating peptide libraries have been developed and described. For example, members of the peptide library can be created by split-synthesis performed on a solid support such as polystyrene or polyacrylamide resin, as described by Lam et al. (1991) Nature 354:82 and PCT publication WO 92/00091. The method disclosed by U.S. Pat. No. 4,833,092 involves the synthesis of peptides in a methodical and predetermined fashion, so that the placement of each library member peptide gives information concerning the synthetic structure of that peptide.
Phage display of peptide libraries has become a powerful tool for rapidly screening and identifying novel ligands of virtually any protein target. Of particular interests are display methods using filamentous bacteriophages. U.S. Pat. No. 5,821,047. This method allows the preparation of libraries as large as 1010–1012 unique peptide members, many orders of magnitude larger than libraries that may be prepared synthetically. In addition to large library sizes, advantages of phage display include ease of library construction (Kunkel mutagenesis), coupling of the binding entity (displayed peptide) to a unique identifier (its DNA sequence), a selection protocol for amplifying rare binding clones in a pool, and the high fidelity of biosynthesis (compared to synthetic methods). Furthermore, rapid and inexpensive selection protocols are available for identifying those library members that bind to a target of interest. However, only natural peptides composed of L-amino acids may be displayed on phage, so the problem of defining three-dimensional structure-activity relationships is more difficult than it might be for a constrained peptidomimetic containing non-naturally occurring amino acids or nonpeptide components.
One possible solution to this problem is to use the structural constraints of a folded protein to present small variable peptide segments. Considerable effort has been devoted to introducing structural constraints into combinatorial peptide libraries so that the member peptides represent more closely their native states. Several protein scaffolds capable of presenting a sequence of interest in a conformationally-restricted fashion have been identified, including minibody structures (Bianchi et al. (1994) J Mol Biol 236:649–659), β sheets, coiled-coil stem structures (Myszka & Chaiken (1994) Biochem 33:2363–2372), zinc-finger domains, cysteine-linked (disulfide) structures, transglutaminase linked structures, cyclic peptides, helical barrels or bundles, leucine zipper motifs (Martin et al. (1994) EMBO J 13:5303–5309), etc.
A number of identified scaffolds have been used in the construction of combinatorial peptide libraries with structural constraints. U.S. Pat. No. 5,824,483 describes a synthetic peptide library containing peptides featuring α-helical conformation and thus capable of forming coiled-coil dimers with each other. McBride et al. (1996) J Mol Biol 259:819–827 describe a synthetic library of cyclic peptides mimicking the anti-tryptic loop region of an identified proteinase inhibitor. WO 00/20574 and U.S. Pat. No. 6,180,343B1 describe fusion constructs using scaffold proteins such as green fluorescent protein (GFP). Several small protein domains have also been proposed as peptide display scaffolds. Nygren & Uhlen (1997) Curr. Opin. Struct. Biol. 7:463–469; Vita et al. (1998) Biopolymers 47:93–100; Vita et al. (1999) Proc. Natl. Acad. Sci. USA 96:13091–13096; Smith et al. (1998) J. Mol. Biol. 277:317–332; Gururaja et al. (2000) Chem. & Biol. 7:515–527; Christmann et al. (1999) Protein Engng. 12:797–806.
Among the identified protein scaffolds, β-turns (hairpins) have been implicated as an important site for molecular recognition in many biologically active peptides. Smith & Pease (1980) CRC Crit Rev Biochem 8:315–300. Thus, peptides containing conformationally-constrained β-turns are particularly desirable. The great majority of the identified β-turn bearing peptides are cyclopeptides which have been generated by the cyclization of a peptide similar to a sequence in the natural substrate. Milner-White (1989) Trends Pharmacol Sci 10:70–74. These cyclopeptides, however, may still retain significant flexibility. For this reason, many studies have attempted to introduce rigid, nonpeptide compounds which mimic the β-turn. Peptides with such nonpeptide β-turn mimic provide useful leads for drug discovery. Ball & Alewood (1990) J Mol Recog 3:55–64; WO 94/03494. The structural mechanisms by which β-turns are stabilized, and specific strand registers are selected, continue to be the subject of considerable interest.
Several examples have been reported of disulfide-constrained peptides intended to mimic protein hairpins or as de novo designed hairpins. In many cases the design includes D-cysteines at one or both ends, as it was initially thought that disulfide bond geometry was not compatible with the cross-strand geometry of hairpins. However, there are some examples that do use L-cys. Evidence for structure is lacking in most studies of disulfide-cyclized peptides. Examples listed here are those whose structures have been experimentally determined, or that use no unusual amino acids and have potency close to a larger, hairpin-containing natural protein in a biological assay.
The structure of a hexapeptide (Boc-CL-Aib-AVC-NMe) SEQ ID NO: 11 was determined crystallographically, revealing a type II′ turn and β-sheet geometry. Kane et al. J. Am. Chem. Soc. (1988) 110:1958–1963. An octapeptide with the same cysteine spacing was studied by NMR, and has a similar structure with a turn centered on Pro-Gly. Walse et al. (1996) J. Comput.-Aided Mol. Des. 10:11–22. Peptides of the form Ac-CXPGXC-NMe SEQ ID NO: 12 were evaluated by measurement of disulfide exchange equilibria, which indicated turn preferences between peptides of as much as 1 kcal/mol. Milburn et al. (1987) J. Am. Chem. Soc. 109:4486–4496.
An eleven-residue cyclic peptide, CGVSRQGKPYC, based on the gene 5 protein from M13 is stably structured in aqueous solution, as demonstrated by NMR analysis. The cyclic peptide adopts a structure that is quite similar to the corresponding protein loop. The authors claim that well-defined β-hairpin structure had not been previously reported for any unprotected disulfide-constrained cycle. Rietman et al. (1996) Eur. J. Biochem. 238:706–713. This peptide has a Val-Pro pair at the nonhydrogen-bonded sites nearest to the cysteines.
Disulfide-cyclized peptides from the hairpin region of a rabbit defensin have antibacterial activity exceeding (about 5 to 10-fold) that of the linear analogs. Circular dichroism spectroscopy indicates some non-random structure in phosphate buffer. The more potent peptide (CAGFMRIRGRIHPLCMRR) SEQ ID NO: 13 has a Gly-Pro pair at the nonhydrogen-bonded sites nearest to the cysteines. Thennarasu & Nagaraj (1999) Biochem. Biophys. Res. Commun. 254:281–283.
Several peptides from the loops of domain 1 of human CD4 have been studied in Zhang et al. (1996) Nature Biotechnology 14:472–475; Zhang et al. (1997) Nature Biotechnology 15:150–154. In addition to a disulfide constraint, the authors have added exocyclic aromatic amino acids to the peptide termini. No evidence for structure is given, but one cyclic peptide was reported to antagonize both normal CD4 interactions and those involved in CD4-mediated cell entry by HUV.
Few examples exist of small peptides that form a stable tertiary structure without assistance from disulfide bonds or metal ions. Most natural peptides encompassing hairpins are mainly devoid of structure in water or form aggregates. Ramirez-Alvarado et al. (1997) Protein Sci. 6:162–147. A hairpin peptide derived from the B1 domain (the 41–56 residue fragment) of protein G (GB1) has been reported to form a well-populated hairpin (about 50%) in water. Blanco et al. (1994) Nat. Struct. Biol. 1:584–590. The GB1 hairpin has four threonine residues at hydrogen-bonded sites in the strands, including one Thr-Thr cross-strand pair. This is generally believed to be an unfavorable pairing. In addition, there are Trp-Val and Tyr-Phe pairs at adjacent nonhydrogen-bonded sites that might interact to form a small hydrophobic core.
Analysis of hairpin sequences in crystal structures has allowed the de novo design of a series of β-hairpin peptides based on the BH8 peptide. Ramirez-Alvarado et al. (1996) Nat. Struct. Biol. 3:604–612. The target structure was a type I′ turn flanked by three-residue strands. Arg-Gly sequences were added to the ends to improve solubility. One peptide was partially folded into a hairpin conformation (about 30%) as determined by NMR. The importance of inter-strand side chain-side chain interactions was indicated by replacement of certain strand residues with alanine. None of the alanine-substituted peptides showed any tendency to form a hairpin. The same authors reported a second series of experiments in which position i+1 of the turn was varied. Ramirez-Alvarado et al. (1997) J. Mol. Biol. 273:898–912. No peptide was more structured than the original sequence with Asn in the turn. A review describing this work suggested that adding Glu-Lys pairs to the termini of the model peptide may help to stabilize the hairpin. Ramirez-Alvarado et al. (1999) Bioorg. Med. Chem. 7:93–103.
A peptide comprising the N-terminal 17 residues of the globular protein ubiquitin has been shown to form a native-like hairpin in both aqueous methanol and water, albeit at low apparent population. Zerella et al. (1999) Protein Sci. 8:1320–1331. A recent study, Zerella et al. (2000) Protein Sci. 9:2142–2150, focused on the contributions to the stability of the isolated peptides by residues within the turn region. The data indicated that in a peptide where Thr at position 9 was replaced by Asp, U(1-17)T9D, the native conformation was stabilized significantly over that of the wild type sequence. The estimated population of the folded hairpin was only 64%. Moreover, as the authors noted, the structure of the folded state of U(1-17)T9D may be more dynamic than indicated by the final ensemble. The reason for the greater stability upon substitution of the turn residue remains uncertain.
It is an object of the present invention to provide a simple model system for displaying small peptides with stable hairpin structure and methods of using such a model system in constructing and screening constrained peptide libraries useful in biological and therapeutic applications.